JP2003520195A - Treatment of refractory human tumors with epidermal growth factor receptor antagonists - Google Patents

Abstract

  (57) [Summary] A method of inhibiting the growth of a refractory tumor stimulated by a ligand of epidermal growth factor in a human patient comprises treating the human patient with an effective amount of an epidermal growth factor receptor antagonist.

Description

Detailed Description of the Invention

[0001] Background of the Invention: Cancer is the leading cause of 2 of the following the heart attack death in the United States of America. Significant advances have been made in the development of new treatments for this devastating disease. Much of that progress is due to a good understanding of cells in both normal and cancer cells. Normal cells proliferate due to highly regulated activation of growth factor receptors by their respective ligands. An example of such a receptor is the growth factor receptor tyrosine kinase.

Cancer cells also proliferate by activation of growth factor receptors, but lose the careful control of normal growth. Loss of control can be caused by many factors, such as overexpression of growth factors and / or receptors, and the voluntary activation of biochemical pathways regulated by growth factors. Some examples of receptors associated with tumorigenesis include epidermal growth factor (EGFR), platelet-derived growth factor (PDGFR), insulin-like growth factor (IGFR), nerve growth factor (NGF).
R) and fibroblast growth factor (FGF).

Members of the epidermal growth factor (EGF) receptor family are particularly important growth factor receptor tyrosine kinases associated with tumorigenesis of epithelial cells. Should be discovered
The first member of the EGF receptor family was a glycoprotein with an apparent molecular weight of approximately 165 kD. Mendelsohn etc. This glycoprotein described by U.S. Pat. No. 4,943,533 is used as an EGF receptor (EGFR)
Also known as HER1).

EGFR is overexpressed on many types of epidermoid tumor cells. EGF and transforming growth factor alpha (TGF-alpha) are two known ligands for EGFR. Examples of tumors that express the EGF receptor include glioblastoma and cancers of the lung, breast, head and neck, and bladder. Amplification and / or overexpression of the EGF receptor on the membrane of tumor cells is associated with poor prognosis.

Treatment of cancer conventionally includes chemotherapy or radiation therapy. Some examples of chemotherapeutic agents include doxorubicin, cisplastin and taxol. Radiation is either from an external beam or a source located inside the patient, ie brachytherapy. Another type of treatment involves antagonists of growth factors or growth factor receptors associated with cell proliferation. Such antagonists neutralize the activity of growth factors or receptors and inhibit the growth of tumors that express the receptor.

For example, US Pat. No. 4,943,533 describes a murine monoclonal antibody designated 225 that binds to the EGF receptor. This patent is assigned to the University of California and licensed exclusively to ImClone Systems, Incorporated.
The 225 antibody is capable of inhibiting the growth of cultured EGFR-expressing tumor lines, and growth of those tumors in vivo when grown as xenografts in nude mice. See Masui et al., Cancer Res. 44, 5592-5598 (1986).

A drawback of using murine monoclonal antibodies in human therapy is the potential for human anti-mouse antibody (HAMA) responses due to the presence of mouse Ig sequences. This drawback is
It can be minimized by replacing the complete constant region of a murine (or other non-human mammalian) antibody with a human constant region. Replacement of the constant region of a murine antibody with human sequences is commonly referred to as chimerization. The chimerization step can also be made more effective by replacing the framework variable region of the murine antibody with its corresponding human sequence. The skeletal variable region is a variable region of an antibody other than the hypervariable region. Hypervariable regions are also complementarity-determining regions (CDRs)
Known as.

Replacement of the constant and framework variable regions with human sequences is commonly referred to as humanizing. Humanized antibodies are less immunogenic (ie, elicit a lower HAMA response) when more murine sequences are replaced by human sequences. Unfortunately, if more regions of the murine antibody are replaced by human sequences, both cost and effort will increase.

Replacement of non-human constant regions with human constant regions does not appear to affect antibody activity. For example, Prewett et al., Described the chimeric forms of anti-EGFR255 discussed above.
We have reported inhibition of tumor progression in well-established prostate tumor xenografts in mice by monoclonal antibodies. This chimeric form is called c225; Jour
nal of Immunotherapy 19, 419-427 (1997).

Another approach to reduce the immunogenicity of antibodies is the use of antibody fragments. For example About-Pirak, Journal of the National Cancer Institute 8
The article by 0, 1605-1611 (1988) compares the anti-tumor effect of an anti-EGF receptor antibody called 108.4 with a fragment of that antibody. This tumor model is based on KB cells as xenografts in nude mice. KB cells are derived from human oral epidermoid carcinoma and express high levels of EGF receptor.

Aboud-Pirak et al. Found that both the antibody and the divalent F (ab ') ₂ fragment prolong tumor growth in vivo, whereas the F (ab') ₂ fragment was almost ineffective. It was However, monovalent Fab fragments of antibodies, which retain the ability to bind cell-associated receptors, delay tumor growth. Attempts have also been made to improve cancer treatment by combining some of the above techniques. For example, Baselga and others are Journal of the National Cancer Institut
e 85, 1327-1333 (1993), reported the anti-tumor effect of the chemotherapeutic agent dexorubicin together with anti-EGFR monoclonal antibody.

On the other hand, attempts have been made to increase the sensitivity of cancer cells to radiation by combining radiation with adjuvants. For example, Bonnen, US Patent 4.846.782.
The issue reports an increased sensitivity of human cancers to radiation when the radiation is combined with interferon. Snelling et al. Showed a slight improvement in radiation treatment of patients with astrocytomas with anaplastic foci when radiation was combined with ¹²⁵ I radiolabeled anti-EGFR monoclonal antibody in Phase II clinical trials. Reporting. See Hybridoma 14, 111-114 (1995).

Similarly, Balaban et al. Of an anti-EGFR monoclonal antibody that sensitizes human squamous cancer xenografts in mice to radiation when administered with an anti-EGFR antibody called LA22 before radiation treatment. Reporting ability. Biochimica et Biophy
See sica Acta 1314, 147-156 (1996). Saleh et al. Also report good tumor control in vitro and in mice when radiation therapy is enhanced by anti-EGFR monoclonal antibodies. Saleh et al.
It can lead to new, combined and characteristic RT / Mab therapies. ”See Abstract 4197 in the American Association for Cancer Research 37,612 (1996) newsletter.

Despite the above treatments for controlling cancer, there is no particular direction for treatment of tumors that are inaccessible to conventional chemotherapy and radiation. Refractory tumors lead to rapid disease progression, usually with a poor prognosis. Currently, it can hardly be done for patients with tumors that are beyond the control of conventional cancer treatments. Based on the foregoing, there is a need for improved methods for treating refractory tumors in humans.

[0015] SUMMARY OF THE INVENTION: The person this and other objects such as will be apparent to skilled in the art, a method of inhibiting the proliferation of flame冶性tumors that are stimulated by a ligand of epidermal growth factor receptor in human patients (EGFR) Achieved by offering. The method comprises treating a human patient with an effective amount of an EGFR / HER1 antagonist. In another embodiment, the method of the present invention comprises treating a human patient with an effective amount of a combination of an EGFR / HER1 antagonist and a chemotherapeutic agent. In yet another embodiment, the method comprises treating a human patient with a combination of the method of the invention, an effective amount of an EGFR / HER1 antagonist and radiation.

Specific Description of the Invention: The present invention provides improved methods for treating refractory tumors, especially refractory malignancies, in human patients with refractory cancer.

Refractory tumors : Refractory tumors include tumors that are not treated or are resistant to treatment with chemotherapeutic agents alone, radiation alone, or a combination thereof. In the context of the present invention, refractory tumors also appear to be inhibited by treatment with chemotherapeutic agents and / or radiation, but up to 5 years after discontinuing treatment, sometimes 10 years or more. It includes tumors that recur over time. The types of refractory tumors that can be treated according to the present invention are any refractory tumor that is not stimulated by the ligand of EGFR. Some examples of ligands that stimulate EGFR include EGF and TGF-α.

The EGFR family of receptors includes the EGFR referred to in the literature as HER1. As used herein, EGFR refers to a specific member of the EGFR family of receptors called EGFR / HER1. Refractory tumors that can be treated by the present invention are endogenous tumors native to human patients.
Those tumors are more difficult to treat than exogenous human tumor xenografts that are treated in animals. For example, Prewett et al., Journal of Immunotherapy 19, 419.
-427 (1997).

Some examples of refractory tumors include cancer, gliomas, sarcomas, adenocarcinomas, adenosarcomas and adenomas. Such tumors are present in virtually all parts of the human body, including all organs. Tumors include, for example, breast, heart, lung, small intestine, colon,
Spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow,
It can be present in blood, thymus, uterus, testis, neck and liver. Tumors can express EGFR at normal levels, or they can overexpress EGFR, for example at levels that are at least 10,100 or 1000 times normal. Some tumors that overexpress EGFR include breast, lung, colon, kidney, bladder,
It includes squamous cell carcinomas of the head and neck, especially the head and neck, ovary, prostate and brain.

EGFR / HER1 Antagonists : Refractory tumors of the invention can be treated with EGFR / HER1 antagonists. For the purposes of the present invention, an EGFR / HER1 antagonist is an EGFR / HER1 ligand-mediated EGFR / HE
Any substance that inhibits the stimulation of R1. Inhibition of such stimuli is EGFR / HE
Inhibits the growth of cells expressing R1.

The growth of refractory tumors is sufficiently inhibited in patients to prevent or slow the progression (ie, growth, invasion, metastasis and / or recurrence) of cancer. EGFR of the present invention
Antagonists can be cell growth regulated or inhibit the growth of refractory tumors. Preferably, the ERGR antagonist is cytostatic or
Or destroy the tumor.

A particular mechanism of inhibition is not involved when functioning in the present invention. Nevertheless, EGFR tyrosine kinases are generally activated by the phenomenon of phosphorylation. Accordingly, phosphorylation assays are useful in predicting antagonists useful in the present invention. Some useful assays for EGFR tyrosine kinase activity are described in Panek et al., Journal of Pharmacology and Experimental Therapeut.
ics 283, 1433-1444 (1997), and Batley et al., Life Sciences 62, 143-150 (1
998). The description of those assays is incorporated herein by reference.

EGFR / HER1 antagonists include biological or small molecules. Biological molecules include all lipids and polymers of monosaccharides, amino acids and nucleotides with over 450 molecules. Thus, biological molecules include, for example, oligosaccharides and polysaccharides; oligopeptides, polypeptides and proteins; and oligonucleotides and polynucleotides. Oligonucleotides and polynucleotides are
For example, DNA and RNA are included.

Biological molecules further include derivatives of any of the above molecules. For example, derivatives of biological molecules include lipid and glycosylated derivatives of oligopeptides, polypeptides, peptides and proteins. Derivatives of biological molecules are also
Included are lipid derivatives of oligosaccharides and polysaccharides, such as lipopolysaccharide. Most typically,
A biological molecule is an antibody, or a functional equivalent of an antibody. A functional equivalent of an antibody has binding properties that correspond to those of the antibody, and inhibits the growth of cells expressing EGFR. Such functional equivalents include, for example, chimerized, humanized and single chain antibodies, and fragments thereof.

Functional equivalents of antibodies also include polypeptides having amino acid sequences that are substantially the same as the amino acids of the variable or hypervariable regions of the antibodies of this invention. Amino acid sequences that are substantially the same as another sequence, but that differ from the other sequence by one or more substitutions, additions, and / or deletions are considered to be equivalent sequences. Preferably, less than 50%, more preferably less than 25%, and even more preferably less than 10% of the number of amino acid residues in the sequence are replaced, added or deleted from the protein.

The functional equivalent of the antibody is preferably a chimerized or humanized antibody. A chimerized antibody comprises the variable region of a non-human antibody and the constant region of a human antibody. Humanized antibodies are the hypervariable regions of non-human antibodies, such as the skeletal variable regions and the constant regions of humanized antibodies are those of human antibodies. For the purposes of the present invention, suitable variable and hypervariable regions of non-human antibodies can be derived from antibodies produced by any non-human mammal for which monoclonal antibodies are produced. Suitable examples of non-human mammals include, for example, rabbits, rats, mice,
Includes horses, goats or primates. Mice are preferred.

Functional Equivalents Also included are fragments of antibodies that have binding properties that are similar to or comparable to the binding properties of the intact antibody. A suitable fragment of an antibody is a hypervariable (ie, complementary) that binds EGFR tyrosine kinase specifically and with sufficient affinity to inhibit the growth of cells expressing such receptor. Includes any fragment comprising a sufficient portion of the sex determining region. Such fragments can include, for example, one or both Fab fragments or F (ab ') ₂ fragments. Preferably, the antibody fragment comprises all 6 complementarity determining regions of the complete antibody, provided that it is less than all such regions, eg a functional fragment comprising 3, 4 or 5 CDRs. Are also included.

Preferred fragments are single chain antibodies or Fv fragments. Single chain antibodies are polypeptides that comprise at least the variable region of the H chain of an antibody linked to the variable region of the L chain, with or without an interconnecting linker. Therefore, F
The v fragment comprises the complete antibody binding site. The chains can be produced in bacteria or eukaryotic cells. Antibodies and functional equivalents include antibodies of any class of immunoglobulin, such as IgG, I.
It can be a member of gM, IgM, IgA, IgD or IgE, and their subclasses. Preferred antibodies are members of the IgG1 subclass. Functional equivalents can also be equivalents of any combination of the above classes and subclasses.

Antibodies may be produced from the desired receptor by methods well known in the art. Receptors are commercially available or can be isolated by well known methods. For example, a method for isolating and purifying EGFR is found in Spada, US Pat. No. 5,646,153 (page 40, left column, starting at line 55). S
The method for isolating and purifying the EGFR described in the pada patent is incorporated herein by reference.

Methods for producing monoclonal antibodies are described in Kohler and Milstein, Nature.
256, 495-497 (1975), and Campbell, “Monoclonal Antibody Technology, The.
Production and Characterization of Rodent and Human Hybridoma ”in Burd
on et al., Eds, Laboratory Techniques in biochemistry and Molecular Biolo
gy, Volume 13, Elsevier Science Publishers, Amsterdam (1985). Huse et al., Science 246, 1275-1281 (1989)
Recombinant DNA methods described by G., are also suitable.

Briefly, to produce a monoclonal antibody, a host mammal is inoculated with the receptor or fragment thereof, as described above, and then optionally boosted. To be useful, the receptor fragment should contain sufficient amino acid residues to define the epitope of the molecule being detected. If the fragment is too short to be immunogenic, it can be conjugated to a carrier molecule. Some suitable carrier molecules are keyhole limpets (limpets)
Includes hemocyanin and bovine serum albumin. Bonding can be done by methods known in the art. One such method is to combine the cysteine residues on the carrier molecule with the cysteine residues of the fragment.

Spleens are harvested from their inoculated mammals a few days after the final booster immunization. The cell suspension from the spleen is fused by the tumor cells. The resulting hybridoma cells that express the antibody are isolated, expanded, and maintained in culture. Suitable monoclonal antibodies, and growth factor receptor tyrosine kinases for producing them are described, for example, in Upstate Biotechnology, Santa Cruz Biotechnology of
Santa Cruz, California, Transduction Laboratories of lexington, Kentuck
y, R & D Systems Inc of Minneapolis, Minnesota, and Dako Corporation of
Available from Carprinteria, California.

Methods for producing chimeric and humanized antibodies are also known in the art. For example, methods for producing chimeric antibodies are described in Boss (
Celltech) and Cabilly (Genentech). Methods for producing humanized antibodies are described, for example, in Winter.
, US Pat. No. 5,225,539.

A preferred method for humanizing antibodies is called CDR-grafting. In CDR-grafting, a region of the mouse antibody that is directly involved in binding to the antigen, the complementarity determining region or CDR, is a human variable region to create a "reshaped human" variable region. Ported inside. These fully humanized variable regions are then ligated to human constant regions to create fully "fully humanized" antibodies.

In order to create fully humanized antibodies that bind well to antigen it is convenient to carefully plan the reshaped human variable region. The human variable region into which the CDRs will be grafted should be carefully selected, and it is usually necessary to make a few amino acid changes at critical positions within the human variable region framework region (FR). Is.

For example, a reshaped human variable region may have up to 10 amino acid changes in the FRs of the selected human L chain variable region, and as many as 12 in the FRs of the selected human H chain variable region. Amino acid changes of The DNA sequences encoding the reshaped human H chain and L chain variable region genes are ligated to the DNA sequences encoding human H chain and L chain constant region genes, preferably γ1 and κ, respectively. The reshaped humanized antibody is then expressed in mammalian cells and its affinity for its target is compared to that of its corresponding murine and chimeric antibody.

Methods for selecting which humanized antibody residues to substitute and for making substitutions are well known in the art. For example, Co., Natur
e 351, 501-502 (1992); Queen et al., Proc. Natl. Acad. Sci. 86, 10029-100.
30 (1989), and Rodrigues et al., Int. J. Cancer, Supplement 7, 45-50 (1992).
) checking. Methods for humanizing and reshaping the 225 anti-EGFR monoclonal antibody are described in Goldstein et al. PCT application WO 96/40210.
This method humanizes antibodies to other growth factor receptor tyrosine kinases,
It can then be modeled for reshaping.

Methods for producing single chain antibodies are also known in the art. Some suitable examples are Wels et al., European Patent Application No. 502812 and Int. J. Can.
cer 60, 137-144 (1995). Other methods for producing the functional equivalents described above are PCT application WO93 / 21319, European patent application 239,400, PCT application WO89 / 09622, European patent application 33.
No. 8,745, US Pat. No. 5,658,570, US Pat. No. 5,693,780 and European patent applications are disclosed in EP 332,424.

Preferred EGFR antibodies are chimeric, humanized and single chain antibodies derived from the murine antibody designated 225, described in US Pat. No. 4,943,533. The patent is assigned to the University of California and ImClone System Incor
Exclusively licensed to porated.

The 225 antibody is able to inhibit the growth of cultured EGFR / HER1-expressing tumor cells in vitro and when grown as xenografts in nude mice in vivo. See Masui et al., Cancer Res. 44,5592-5598 (1986). More recently, treatment methods combining 225 and doxorubicin or cisplatin have shown therapeutic synergism on several well-established human xenograft models in mice. Basalga et al., J. Natl. Cancer Inst. 85, 1327-1333 (1
See 993).

In one aspect of the invention, the refractory head and neck squamous cell carcinoma is EGFR / HER
Treated with a combination of 1 antagonist (chimeric anti-EGFR monoclonal antibody, C225) and cisplatin. Those patients were unable to achieve conventional treatment with radiation alone, chemotherapy alone or a combination thereof. The EGFR / HER1 antagonist inhibited the growth of refractory tumors.

Chimeric, humanized, and single chain antibodies derived from murine antibody 225 can be produced from the 225 antibody available from the ATCC. On the other hand, the various fragments required to prepare the chimerized, humanized, and single chain 225 antibody are described by Wels et al., Int. J. Cancer 60, 137-144 (1995 ). The chimerized 225 antibody (c225) can be produced according to the method described above. The humanized 225 antibody can be prepared according to the method described in Example IV of PCT application WO 96/40210, which is incorporated herein by reference. Single strand 2
The 25 antibody (Fv225) can be prepared according to the methods described in Wels et al., Int. J. Cancer 60, 137-144 (1995) and European Patent Application No. 502,812.

The hypervariable variable (CDR) regions of the L and H chains are reproduced below. The amino acid sequence is shown below the nucleotide sequence.

[Chemical 1]

In addition to the biological molecules discussed above, the antagonists useful in the present invention can also be small molecules. Any molecule that is not a biological molecule is considered herein to be a small molecule. Some examples of small molecules include organic compounds, organometallic compounds, salts of organic and organometallic compounds, sugars, amino acids and nucleotides. Small molecules also include molecules that are considered biological molecules, provided their molecular weight is no higher than 450. Thus, small molecules can be lipids, oligosaccharides, oligopeptides and oligonucleotides, and their derivatives, having a molecular weight of 450 or less.

It is emphasized that small molecules can have any molecular weight. They are typically referred to as small molecules because they typically have a molecular weight of 450 or less.
Small molecules include compounds found in nature and synthetic compounds. Preferably, the small molecule inhibits the growth of refractory tumor cells that express the EGFR / HER1 tyrosine kinase.

Many small molecules have been described as useful for inhibiting EGFR. For example, Spada et al., US Pat. No. 5,656,655 disclose heteroaryl compounds to styryl substitutions that inhibit EGFR. A heteroaryl group is a monocyclic ring having 1 or 2 heteroatoms, or a bicyclic ring having 1 to about 4 heteroatoms, the compound being optionally substituted or polysubstituted. To be done. US Patent No. 5,65
The compounds disclosed in 6,655 are incorporated herein by reference.

Spada et al., US Pat. No. 5,646,153 disclose bisamino and / or bicyclic arylheteroaryl, carbocyclic and heterocarbocyclic compounds that inhibit EGFR. The compounds disclosed in US Pat. No. 5,646,153 are incorporated herein by reference. Bridges et al., US Pat. No. 5,679,683, discloses tricyclic pyrimidine compounds that inhibit EGFR. The compound is described on page 2, left column, lines 35 to 4, left column,
Fused heterocyclic pyrimidine derivatives described in line 6. The description of those compounds on page 2, left column, line 34 to page 4, left column, line 6 is incorporated herein by reference.

Barker, US Pat. No. 5,616,582 discloses quinazoline derivatives having receptor tyrosine kinase inhibitory activity. The compounds disclosed in US Pat. No. 5,616,582 are incorporated herein by reference. Fry et al., Science 265, 1093-1095 (1994) discloses compounds having structures that inhibit EGFR. Its structure is shown in FIG. The compounds shown in Figure 1 of Fry et al. Are incorporated herein by reference.

Osherov and the like are tyrphostins that inhibit EGFR / HER1 and HER2
Will be disclosed. Documents such as Osherov and especially the compounds disclosed in Tables I, II, III and IV are incorporated herein by reference. Levitzki et al., US Pat. No. 5,196,446 discloses heteroarylethenediyl or heteroarylethenediylaryl compounds that inhibit EGFR.
The compounds disclosed in U.S. Pat. No. 5,196,446, page 1, right column, line 42 to page 2, left column, line 40 are incorporated herein by reference.

Panek et al., Journal of Pharmacology and Experimental Therapeuticis 283
, 1433-1444 (1997) inhibits the EGFR, PDGFR and FGFR families of receptors, P
Disclosed is the compound identified as D166285. PD16628 is 6- (2,6-dichlorophenyl) -2- (4- (2-diethylaminoethoxy) phenylamino) -8-methyl-8H-pyrido (2,3) having the structure shown in Figure 1 on page 1436. −
d) Identified as pyrimidin-7-one. The compounds described in Figure 1 on page 1436 of Panek et al. Are incorporated herein by reference.

Administration of EGFR / HER1 Antagonists : The present invention encompasses administering an effective amount of EGFR / HER1 antagonists to a human patient. Administration of EGFR / HER1 antagonists can be accomplished by a variety of means, including systemically by the parenteral and enteral routes. For example, the EGFR / HER1 antagonists of the present invention can be readily administered intravenously (eg, by intravenous injection), which is the preferred route of delivery. Intravenous administration may be accomplished by contacting the EGFR / HER1 antagonist with a suitable pharmaceutical carrier (vehicle) or excipient, as will be appreciated by those in the art. EGFR / HER1 antagonists can be administered with adjuvants such as BDG, immune system stimulants and chemotherapeutic agents.

EGFR / HER1 antagonists that are small molecule or biological agents are administered as described in Spada, US Pat. No. 5,646,153, page 56, left column, page 47, left column, line 67. Can be done. This description of administering small molecules is incorporated herein by reference. The EGFR / HER1 antagonists of the present invention, when administered to human patients in effective amounts,
Significantly inhibits the growth of refractory tumor cells. As used herein, an effective amount is an amount effective to achieve the particular result of inhibition of the growth of a refractory tumor.

Preferably, the EGFR / HER1 antagonist is provided to the tumor in an amount that inhibits tumor growth without interfering with normal tissue growth. Most preferably, the EGFR / HER1 antagonist inhibits tumor growth without severe side effects. Some serious side effects include myelosuppression, anemia and infection.

The optimal dose of EGFR / HER1, the antibody and the functional equivalent of the antibody, will depend on many parameters, such as age, sex, weight, severity of the condition being treated, the antibody administered, and the route of administration. Based on this, it can be determined by a doctor. Generally, serum concentrations of polypeptides and antibodies that allow saturation of the target receptor are desired. For example, about 0.1n
Concentrations above M are usually sufficient. For example, a dose of C225 of 100 mg / m ² provides a serum concentration of about 20 nM for about 8 days. As a rough guideline, antibody doses may be given weekly in amounts of 10-300 mg / m ² . Equivalent doses of antibody fragments should be used at more frequent intervals to maintain serum levels above the concentration that allows receptor saturation.

Combination Therapy : In one preferred embodiment, the refractory tumor may be treated with an effective amount of an EGFR / HER1 antagonist together with a chemotherapeutic agent, radiation or a combination thereof. Examples of chemotherapeutic agents or chemotherapeutic agents are alkylating agents such as nitrogen mustards, ethyleneimine compounds, alkylsulfonates and other compounds having an alkylating action such as nitrosoureas, cisplatin and dacarbazine; antimetabolites such as folic acid. , Purine or pyrimidine antagonists; mitotic inhibitors such as vinca plant alkaloids and podophyllotoxins; cytotoxic antibiotics and camptothecin derivatives.

Camptothecin derivatives include, for example, camptothecin, warethylcamptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), 9-aminocamptothecin, 10,11-methylenedioxy-camptothecin (MDCPT) and topotecan. . Such camptothecin derivatives are also 7-ethyl-disclosed in US Pat. No. 5,604,233, the disclosure of which is incorporated herein by reference.
Also included is a lactone stable formulation of camptothecin.

The present invention provides highly lipophilic camptothecin derivatives, such as 10,11-methylenedioxy-camptothecin, 10,11-ethylenedioxy-camptothecin, 9-ethyl-camptothecin, 7-ethyl-10-hydroxy-camptothecin, 9-methyl-camptothecin, 9-chloro-10,11-methylenedioxy-camptothecin,
9-chloro-camptothecin, 10-hydroxy-camptothecin, 9,10-dichloro-camptothecin, 10-bromo-camptothecin, 10-chloro-camptothecin, 9-fluoro-camptothecin, 10-methyl-camptothecin, 10-fluoro-camptothecin, Includes 9-methoxy-camptothecin, 9-chloro-7-ethyl-camptothecin and 11-fluoro-camptothecin. Such highly lipophilic camptothecin derivatives are disclosed in US Pat. No. 5,880,133, which is incorporated herein by reference.

Examples of the water-soluble camptothecin derivative include CPT-11, 11-hydroxy-7-alkoxy-camptothecin, 11-hydroxy-7-methoxycamptothecin (11,
7-HMCPT) and 11-hydroxy-7-ethylcamptothecin (11,7-HECPT)
, 7-dimethylaminomethylene-10,11-methylenedioxy-20 (R, S) -camptothecin, 7-dimethylaminomethylene-10,11-methyleneoxy-20 (S)
-Camptothecin, 7-dimethylaminomethylene-10,11-ethylenedioxy-
20 (R, S) -camptothecin and water-soluble analogs of camptothecin known as 7-morpholinomethylene-10,11-ethylenedioxy-20 (S) -camptothecin. Such water-soluble camptothecin derivatives are disclosed in US Pat. No. 5,559,235, the disclosure of which is incorporated herein by reference.

Preferred chemotherapeutic agents or chemotherapeutic agents are amifostine, cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), Doxorubicin (adriamycin), doxorubicin lipo (
Doxil), gemcitabine (gem monkey), daunorubicin lipo (daunoxome), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel, astaxelet, astaxel, astaxel, taxotere, astaxel, taxoterex, aldetaxel, taxotere, astaxel, taxotere Busulfan, carboplatin,

Cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), decarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon α
, Interferon β, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin,
Includes meat ten, pega spargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, test lactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil and combinations thereof.

Administration of chemotherapeutic agents can be accomplished systemically by a variety of means, including parenteral and enteral routes. Preferably, the chemotherapeutic agent is administered intravenously by contacting the chemotherapeutic agent with a suitable pharmaceutical carrier (vehicle) or excipient, as will be appreciated by those in the art. The dose of the chemotherapeutic agent depends on many factors as are well known in the art. Such factors include age, gender, weight,
It includes the severity of the condition being treated, the agent administered, and the route of administration. For example,
Cisplatin may conveniently be administered at a dose of about 100 mg / m ² . However, it should be emphasized that the present invention is not limited to any particular dose.

In yet another embodiment, the refractory tumor can be treated with an effective amount of an EGFR / HER1 antagonist in combination with radiation. The radiation source can be either external or internal to the patient being treated. When the radiation source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the process is called brachytherapy.

Radiation is administered according to well known standard techniques by standard equipment manufactured for this purpose, for example AECL Theraton and Varian Clinac. The dose of radiation will depend on factors as are well known in the art. Such factors include the organs to be treated, the healthy organs in the radiation path that may inadvertently be adversely affected, the patient's tolerance for radiation therapy and the area of the body in need of treatment. The dose will typically be 1-100 Gy and more particularly 2-80 Gy.
Some reported doses are 35 Gy for the spinal cord, 15 Gy for the kidney, 20 Gy for the liver and 65-80 Gy for the prostate. However, it should be emphasized that the present invention is not limited to any particular dose. The dose will be determined by the treating physician according to particular factors in the given circumstances, such as those mentioned above.

The distance between the external radiation source and the point of entry into the patient can be any distance that provides an acceptable balance between target cells to kill and minimal side effects. Typically, the external radiation source is 70-100 cm from the point of entry into the patient. Brachytherapy is generally performed by placing a radiation source on the patient.
Typically, the radiation source is placed about 0-3 cm from the tissue to be treated.
Known techniques include interstitial, intercavity, and superficial brachytherapy. The radioactive species can be implanted permanently or temporarily. Some typical radioactive atoms that have been used for permanent implantation include ¹²⁵ I and radon. Some typical emitting atoms that have been used for temporary implants include radium, cesium-137 and iridium-192. Some additional radioactive atoms that have been used in brachytherapy include Americium-241 and Gold-198.

The radiation source for brachytherapy may be the same as the radiation source mentioned above for external beam radiation therapy. In addition to the factors mentioned above for determining the dose of external beam radiation therapy, the nature of the radioactive atom used is taken into account in the dose determination of brachytherapy.

In a preferred embodiment, when the refractory tumor in a human patient is treated with an EGFR / HER1 antagonist and a chemotherapeutic agent or radiation, or a combination thereof,
There is synergy. In other words, the inhibition of tumor growth by EGFR / HER1 antagonists is enhanced when combined with chemotherapeutic agents or radiation, or a combination thereof. Synergy is obtained by greater inhibition of refractory tumor growth by the combined treatments than would be expected from treatment alone, for example with either EGFR / HER1 antagonists, chemotherapeutic agents or radiation. Preferably, synergism is that EGFR
/ As indicated by unexpected cancer regression from treatment with HER1 antagonists, chemotherapeutic agents or radiation alone.

The EGFR / HER1 antagonist may be before, during or after the initiation of the chemotherapeutic agent or radiation therapy, any combination thereof, ie before and during the initiation of the chemotherapeutic agent and / or radiation therapy, and after, Administered between and after, or before, during and after. For example, EG
When the FR / HER1 antagonist is an antibody, it is typically administered 1-30 days, preferably 3-20 days, more preferably 5-12 days prior to the initiation of radiation and / or chemotherapy.

[0068] Example Example 1. Clinical Trials : In clinical trials, human patients with refractory head and neck squamous cell carcinoma were treated with EG
Treated with a combination of FR / HER1 antagonist (chimeric anti-EGFR monoclonal antibody, C225) and cisplatin. Patients with cisplatin in combination of 100 mg / m ² every three weeks, 100/100, 400/250 or 500/250 mg / m C22 at ² load / maintenance dose
Received 5 weekly infusions. Tumor samples were obtained at baseline, 24 hours after the initial injection and 24 hours before the third injection, to assess tumor EGFR saturation and function.

Immunohistochemistry (IHC) using tumor EGFR saturation with M225 (the murine counterpart of C225) as the first antibody and anti-mouse IgG as the second antibody to detect unoccupied EGFR. It was evaluated by. Activated EGFR (Transduction
Labs) was evaluated by IHC with an antibody specific for Labs) and after clearance of the C225-EGFR complex by measurement of EGFR tyrosine kinase activity on tumor lysates. A dose-dependent increase in receptor saturation was demonstrated with> 70% receptor saturation throughout the 500/250 mg / m ² dose level. Similarly, no significant reduction in EGFR-tyrosine kinase activity has been shown with detectable activity in 67% of patients at the 100/100 mg / m ² dose, suggesting functional saturation.

Adverse events were fever, allergies and skin toxicity manifested as vesicular rashes or nail bed changes that resolved well after cessation of treatment. In 7 evaluable patients, there was one minimum, 5 partial and 1 complete response, as determined by physical examination and experimental values. A complete response was observed in one patient who received prior cisplatin treatment. Partial responses were observed in 5 patients, where 4 had prior chemotherapy and 1 had prior radiation treatment. Minimal response was observed in one patient who received prior radiation treatment. The results are shown in the table, where CR means complete response and PR
Means partial response and MR means minimal response.

[0071]

[Table 1]

Example 2. Clinical Trials : In clinical trials, one human patient with refractory colon cancer was treated with a combination of EGFR / HER1 antagonist (chimeric anti-EGFR monoclonal antibody, C225) and CPT-11. Patients with CPT-11 combined with the 125 mg / m ^2, received injections weekly C225 at a loading dose of 400 mg / m ^2. 69-125 with CPT-11 and the union of ^{mg / m 2 250 mg / m} 2
A maintenance dose of C225 was administered weekly. Clinically, the patient had a complete response. The dosing schedule is summarized in Table 2 below.

[0073]

[Table 2]

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Claims (33)

[Claims] 1. A method for inhibiting the growth of a refractory tumor stimulated by a ligand of epidermal growth factor receptor (EGFR) in a human patient, which comprises the step of administering an effective amount of EGFR / human EGF receptor-1 (HER1) antagonist A method comprising treating the human patient. 2. The method according to claim 1, wherein the antagonist is a monoclonal antibody specific for EGFR / HER1, or a fragment comprising a hypervariable region thereof. 3. The method according to claim 2, wherein the monoclonal antibody is chimerized or humanized. 4. The method of claim 1, wherein the antagonist is a small molecule that specifically binds EGFR / HER1. 5. The method of claim 4, wherein the small molecule inhibits EGFR / HER1 phosphorylation. 6. The method of claim 2, wherein the monoclonal antibody inhibits EGFR / HER1 phosphorylation. 7. The method of claim 1, wherein the refractory tumor has been treated with radiation or chemotherapy, and combinations thereof. 8. The tumor is breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus. The method according to claim 1, which is a tumor of testis, neck, or liver. 9. The method of claim 1, wherein the tumor is squamous cell carcinoma. 10. A method of inhibiting the growth of a refractory tumor stimulated by a ligand for epidermal growth factor receptor (EGFR) in a human patient, wherein the human patient is treated with a combination of an effective amount of EGFR / HER1 antagonist and radiation. A method comprising processing. 11. The method of claim 10, wherein the antagonist is administered prior to the administration of radiation. 12. The method of claim 10, wherein the antagonist is administered during radiation administration. 13. The method of claim 10, wherein the antagonist is administered after radiation administration. 14. The method of claim 10, wherein the antagonist is administered before and during radiation administration. 15. The method of claim 10, wherein the antagonist is administered during and after radiation administration. 16. The method of claim 10, wherein the antagonist is administered before and after radiation administration. 17. The method of claim 10, wherein the antagonist is administered before, during and after radiation administration. 18. The method of claim 10, wherein the radiation source is external to a human patient. 19. The method of claim 10, wherein the radiation is internal to a human patient. 20. The antagonist is a monoclonal antibody.
Method described in 10. 21. The tumor is breast, heart, lung, small intestine, colon, spleen, kidney,
Bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus,
11. The method according to claim 10, which is a tumor of the uterus, testis, neck and liver. 22. A method for inhibiting the growth of a refractory tumor stimulated by a ligand for epidermal growth factor receptor (EGFR) in a human patient, wherein the human patient is treated with an effective amount of an EGFR / HER1 antagonist and a chemotherapeutic agent. A method comprising processing. 23. The method of claim 22, wherein the antagonist is administered prior to treatment with the chemotherapeutic agent. 24. The method of claim 22, wherein the antagonist is administered during treatment with the chemotherapeutic agent. 25. The method of claim 22, wherein the antagonist is administered after treatment with the chemotherapeutic agent. 26. The method of claim 22, wherein the antagonist is administered before and during treatment with the chemotherapeutic agent. 27. The method of claim 22, wherein the antagonist is administered during and after treatment with the chemotherapeutic agent. 28. The method of claim 22, wherein the antagonist is administered before and after treatment with the chemotherapeutic agent. 29. The antagonist prior to treatment with the chemotherapeutic agent,
23. The method of claim 22, which is administered during and after. 30. The chemotherapeutic agent is amifostine, cisplatin, dacarbazine, dactinomycin, mechlorethamine, streptozocin, cyclophosphamide, carmustine, lomustine, doxorubicin, doxorubicin lipo, gemcitabine, daunorubicin, procarbazine, mitomycin, cytarabine, Etoposide, methotrexate, 5-fluorouracil, vinblastine,
Vincristine, bleomycin, paclitaxel, docetaxel, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38
), Decarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon α, interferon β, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, meaton, Pega spargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, test lactone, thioguanine, thiotepa,
23. The method of claim 22, selected from the group consisting of uracil mustard, vinorelbine, chlorambucil and combinations thereof. 31. The method of claim 22, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, doxorubicin, paclitaxel, CPT-11, topotecan, and combinations thereof. 32. The tumor is breast, heart, lung, small intestine, colon, spleen, kidney,
Bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus,
23. The method of claim 22, which is a tumor of the uterus, testis, neck and liver. 33. The method of claim 22, wherein the antagonist is a monoclonal antibody.
The method described.